The present invention relates to encapsulated cells producing antibodies, especially antibodies belonging to the various classes of immunoglobulines; IgM, IgD, IgGs, IgE and IgA, and to the use of such encapsulated cells for implantation in vivo for long term delivery or sustained delivery of antibodies of therapeutic interest.
Systemic delivery of cytostatic or cytotoxic tumor-specific antibodies b y engineered cells grafted to patients may be highly valuable for long-term anticancer surveillance treatments to prevent relapse after a primary treatment such as surgery, chemo- or radiotherapy. Such an approach could also be used for treating severe viral diseases, such as AIDS, if virus-neutralizing antibodies or antibodies toxic for virus-producing cells are delivered. In addition, long-term systemic delivery of antibodies may also be useful for more fundamental purposes such as the development of new animal models of autoimmune diseases in which the humoral response contributes to the development of the illness (Rose, N. R. and Bona, C., Immunol. Today, Vol. 14, 426-430 (1993)). Another possible application involve the development of a new cell ablation technique useful for studying in vivo various differentiation pathways and/or the biological importance of specific cell subsets through the release into the blood stream of cytotoxic antibodies recognizing cell type-specific membrane markers. In this situation, antibodies would kill target cells, for instance after a specific differentiation step, immediately following the appearance of cognate antigens at the surface of the differentiating cells.
It has been recently shown thatxe2x80x94by using retroviral gene transferxe2x80x94several cell types (including skin fibroblasts, myogenic cells, hepatocytes and keratinocytes), amenable to genetic modification and grafting to patients, can produce antibodies retaining the specifity and the affinity of the parental antibody (Noel, D et al., Hum. Gene Ther., Vol. 8, 1219-1229 (1997)). Furthermore, the grafting of engineered myogenic cells allows the systemic delivery of cloned antibodies in mouse for at least several months. Although these observations lend support to the idea that engineering of patients"" cells may be useful for long-term antibody-based gene therapies, several issues potentially limit the clinical application of such a technology. First, such a therapeutical approach would be labor-intensive and time consuming. Second, stable genetic modification of patients"" cells currently utilises ex vivo retroviral infection followed by autologous grafting in order to avoid rejection of non-MHC (MHC=major histocompatibility complex) matched cells b y the immune system. This reduces the versatility of the approach since engineered cells from one individual cannot be used for another. Third, efficient gene transfer and long-term expression of transgenes in cells that can be used in gene therapy protocols are issues that have not yet been completely solved (Crystal, R. G., Science, Vol. 270, 404-410 (1995); Harris, J. D. and Lemoine, N. R., Trends Genet., Vol. 12, 400-405 (1996); Vile, R. G. et al., Mol. Biotechnol., Vol. 5, 139-158 (1996)).
In this context, implantation of engineered cells encapsulated into immunoprotective devices into patients may represent a more versatile and cost-effective approach. On the one hand, it should allow the same batch of non-MHC-matched cells (possibly selected in vitro for optimal antibody expression) to be used for several patients and, on the other hand, implantation of capsules is a very simple surgical operation. In addition, such a technique would also offer the possibility of easy surgical removal of antibody-producing cells in case the treatment needs to be terminated.
For optimal function, the capsule pores must meet two criteria. First, they must be large enough to permit molecules of interest, such as antibodies, to exit and to permit the entry and efficient diffusion of nutrients necessary for cell survival. Second, they must be small enough to prevent the encapsulated cells from leaving the capsules and to prevent entry of host immune system cells.
Encapsulation of cells in permeable structures that allow the release of certain biologically active molecules but protects the cells producing these molecules from the host immune system has met with some success (for a review see Chang, P. L. In Somatic Gene Therapy. P. L. Chang, ed. (CRC Press, Boca Raton), p 203-223 (1995)). Cells that have been genetically modified to produce human growth hormone (hGH) (Tai, I. T. and Sun, A. M., FASEB J. 7, 1061-1069 (1993)) or a secreted form of human adenosine deaminase (Hughes et al., Hum. Gene Ther. 5, 1445-1455 (1994)) have been encapsulated. In both of these studies, cells were encapsulated in poly-L-lysine-alginate microcapsules and the cells were shown to survive for long periods in culture. This was accompanied by long term production of the enzyme or hormone. Further, it was shown that upon transplantation of the microcapsules into mice, the cells remained viable for 1 year and they continued to produce hGH, demonstrating that the capsules protect the transfected cells from destruction by the host immune system. Nevertheless, it was also reported that polylysine-alginate capsules induce an inflammatory response (Pueyo, M. E. et al., J. Biomater. Sci. Polym. Ed., Vol. 5, 197-203 (1993); Vandenbossche, G. M. et al., J. Pharm. Pharmacol., Vol. 45, 115-120 (1993)).
Cell encapsulation has also been reported using other materials. Baby hamster kidney cells genetically modified to produce nerve growth factor have been encapsulated in polyacrylonitrile/vinyl chloride and implanted in rat brain. The encapsulated cells survived for at least 6 months and continued to produce NGF (Winn et al., Proc. Natl.Acad. Sci. USA 91, 2324-2328 (1994) and Deglon et al., Gene Ther., 2, 563 (1995)).
Rat hybridoma cells secreting a mAb directed against murine IL-4 have been encapsulated in alginate and implanted, intraperitoneally and subcutaneously, into mice (Savelkoul, H. F. et al., J. Immunol. Methods, Vol. 170, 185-196 (1994)). However, the levels of antibody delivered in the blood stream declined after 14 days as a consequence of capsule deterioration. Moreover, in this system a 100% incidence of ascite development was observed 30 days post-implantation as a result of cell released from the capsules into the intraperitoneal cavity.
Hepatocytes have successfully been encapsulated in a polyelectrolyte complex of cellulose sulphate and polydimethyldiallyl ammonium (Stange et al., Biomat.Art.Cells and Immob. Biotech. 21, 3443-352 (1993)). More than 90% of the encapsulated hepatocytes retained their viability and in contrast to hepatocytes grown as monolayers, the encapsulated cells showed an increased metabolic activity.
The same encapsulation materials have been used for the encapsulation of antibody producing hybridoma cells (Merten et al. Cytotechnology 7:121-130, 1991).The capsules were prepared from a solution of sodium cellulose sulphate (1.5%) and poly-dimethyl-diallyl-ammoniumchloride (2% solution). The influence of varying encapsulation process parameters on capsule characteristics, cell growth, and monoclonal antibody production were tested and it was demonstrated that encapsulation using sodium cellulose sulphate as polyanion and poly-dimethyl-diallyl-ammonium-chloride as polycation, is a suitable tool for the preparation capsules useful for the cultivation of mammalian cells at high densities.
To summarize of what is known from the state of the art either in vivo implantation of the encapsulated cells producing antibodies for long term delivery and/or sustained release of antibodies for therapy is not described. or even suggested, or implantation of capsules resulted in severe side effects as, e.g., inflammatory responses.
It is, thus, an object of the present invention to provide capsules containing antibody-producing cells, which allow the release of the antibodies from the capsules, and which do not elicit inflammatory response after implantation in a host.
The present invention then inter alia comprises the following, alone or in combination:
Capsules encapsulating antibody-producing cells, said capsules comprising a core containing said cells and a porous capsule wall surrounding said core which is permeable to the antibodies produced by said cells;
capsules as above wherein said porous capsule wall consist of a polyelectrolyte complex formed from counter-charged polyelectrolytes;
capsules as above wherein said porous capsule wall consist of a complex formed from cellulose sulphate and polydimethyidiallylammonium;
capsules as any above wherein said cells have been genetically modified to produce cloned antibodies;
capsules as any above wherein said antibodies belong to the IgA, IgM, IgG, IgD or IgE classes of immunoglobulins;
capsules as any above wherein said antibodies are selected from antibodies that bind to
viral surface markers on the surface of virus infected cells,
cancer cells,
T- or B lymphocytes involved in the pathological effects of auto-immune diseases,
surface markers of parasites, or
circulating antigens with deleterious effects (for example autoantibodies)
and have a cytostatic or cytotoxic effect on said cells, or
wherein said antibodies are selected from antibodies that bind to and block viral receptors necessary for viral infection of cells, or direct neutralizing effect via their binding to viruses or direct neutralizing effect on circulating deleterious antigens;
use of the capsules as any above for the implantation into a living animal body, including a human, for the treatment of diseases or disorders responsive to the antibodies released from said capsules;
use as above for subcutaneous implantation;
use of the capsules as any above for producing a pharmaceutical composition for the treatment of diseases or disorders responsive to the antibodies released from said capsules;
a pharmaceutical composition containing capsules as any above for the treatment of diseases or disorders responsive to the antibodies released in a therapeutically effective amount from said capsules;
a method for the treatment of a disorder or disease responsive to the antibodies produced by encapsulated cells as any above comprising implantation of said capsules and/or of the pharmaceutical composition as above into a living animal body, including a human;
a method as above comprising subcutaneous implantation; and
a method as above wherein a cancer or an auto-immune disease is treated, or infections by parasites or pathogenic viruses are treated or prevented.
According to the present invention, capsules containing cells producing antibodies, which allow the release of the antibodies from the capsules, and which do not elicit inflammatory response after implantation in a host, are provided.
The encapsulated cells according to the invention can be prepared by suspending the cells producing antibodies in an aqueous solution of a polyelectrolyte (e.g. selected from sulphate group-containing polysaccharides or polysaccharide derivatives or of sulphonate group containing synthetic polymers), whereafter the solution in the form of preformed particles is introduced into a precipitation bath containing an aqueous solution of a counter-charged polyelectrolyte (such as for example a polymer with quaternary ammonium groups).
Sulphate group-containing polysaccharides or polysaccharide derivatives includes cellulose sulphate, cellulose acetate sulphate, carboxymethylcellulose sulphate, dextran sulphate or starch sulphate in the form of a salt, especially a sodium salt. The sulphonate group-containing synthetic polymer can be a polystyrene sulphonate salt, preferably a sodium salt.
Polymers with quaternary ammonium groups includes polydimethyldiallylammonium or polyvinylbenzyl-trimethylammonium, in the form of a salt thereof, preferably a chloride salt.
In a preferred embodiment of the invention the cells producing antibodies are encapsulated in a complex consisting of a complex formed from cellulose sulphate and polydimethyidiallyl-ammonium.
Methods for the preparation of the cellulose sulphate capsules used for the preparation of the capsules according to the invention has been thoroughly described in DE A1 40 21 050. Also the synthesis of the cellulose sulphate has been described in this patent application. Methods for a comprehensive characterisation of cellulose sulphate capsules have been extensively dealt with in H. Dautzenberg et al., Biomat., Art. Cells and Immob. Biotech., Vol. 21, 399-405 (1993). Cellulose sulphate capsules and their preparation have also been described in GB 2 135 954. The properties of the cellulose capsules, i.e. the size, the pore size, wall thickness and mechanical properties depend upon several factors such as for example physical circumstances whereunder the capsules have been prepared, viscosity of precipitation bath, its ion strength, temperature, rapidity of addition of celVcellulose sulphate suspension, cnstitution of cellulose sulphate, as well as other parameters described by the Dautzenberg group.
The capsules according to the invention can be prepared by suspending the cells producing antibodies in a solution containing 0.5-50%, preferably 0.5-5%, sodium cellulose sulphate and 5% fetal calf serum in an aqueous solution, preferably a buffer. This suspension is then dropped by a dispensing system (e.g. air-jet system or piezoelectric system) into a precipitation bath containinga stirred solution of 0.5%-50%, preferably 0.5-10% polydimethyl-diallylammonium chloride in an aqueous solution, preferably a buffer. Capsule formation occurs within milliseconds and the capsules containing cells are kept in the precipitation bath for 30 seconds to 5 minutes and then washed. The rapidity of this method ensures that the cells are not unduly stressed during the whole procedure (Stange et al., Biomat. Art. Cells and Immob. Biotech. 21. 343-352 (1993)).
The capsules according to the invention have a variable diameter between 0.01 and 5 mm, but are preferably between 0.1 and 3 mm. Consequently, capsules can be made to contain a variable number of cells. Using the encapsulation process according to the invention, up to 1010, but preferably 103-107 cells producing antibodies can be encapsulated in the polyelectrolyte complex.
Capsules composed of cellulose sulphate and polydimethyldiallyl ammonium have excellent mechanical properties and can be manufactured to consistent diameter and pore size.
The encapsulated cells can be cultivated in a normal cell culture medium (the nature of which depends on the encapsulated cells) at standard conditions of humidity, temperature and CO2 concentration. During this culture period production of antibodies from the capsules into the cell culture medium can be demonstrated with either Western Blot or Elisa technology using specific antigens and can furtheron be quantitated using second antibodies conjugated to fluorogenic dyes.
After a suitable period in culture (normally not less than 1 hour and not exceeding 30 days), the cell containing capsules can be surgically implanted either directly, or by injection using a syringe into various areas of the body.
The antibodies produced by the encapsulated cells according to the invention can be based on any immunglobulin class useful for therapy, including but not limited to genetically modified antibodies.
The encapsulated cells according to the invention can be cells taken from patients or from any other source, including human and animal cells, that have been genetically modified for the production of cloned antibodies.
The encapsulated cells and capsules, respectively, are especially used for the implantation into a living animal body, including a human, for the treatment of diseases or disorders responsive to the antibodies released from said capsules. After implantation of capsules into an animal body intraperitoneally and subcutaneously it has been found that the capsules, especially cellulose sulphate capsules, offer an obvious advantage with respect to mechanical resistance over, for instance, alginate capsules since there are found intact as long as 10 months post-implantation regardless of whether they are implanted subcutaneously or intraperitoneally.
Additionally, it has been observed that subcutaneous and intraperitoneal implantations of cell-containing cellulose sulphate capsules revealed differences with respect to at least two points. First, the amount of antibody released in the bloodstream was markedly higher in the former situation. A very likely explanation for this difference resides in the fact that capsules are rapidly vascularized when implanted subcutaneously and are not vascularized at all when implanted intraperitoneally. The beneficial effect of vascularization might be two-fold, firstly facilitating antibody uptake by blood and, secondly, ensuring a better supply of nutrients favoring cell survival since viability of cells within intraperitoneally implanted capsules was reproducibly observed to be lower. In addition to extensive vascularization, which showed no significant alteration over the 10 months of the follow-up, the clustering of cells within a connective pouch after subcutaneous implantation would allow removal of capsules through an easy onestep surgical ablation of the whole neorgan should this prove necessary. Finally, it is important to underline that development of isolating fibrosis around implanted cellulose sulphate capsules is not systematic. This observation contrasts with what has been reported in the case of alginate-poly(L)-alginate microcapsules around which a host reaction with fibrosis developed probably as a result of potent macrophage activation by the encapsulating polymer (Pueyo, M. E, et al., J. Biomater Sci. Polym. Ed., Vol. 5, 197-203 (1993)).
Anti-idiotypic response are often observed in patients repeatedly treated with high doses of purified monoclonal antibodies and can sometimes neutralize the effects of the treatment (Isaacs, J. D., Semin. lmmunol., Vol. 2, 449-456 (1990)). Moreover, the mode of administration of antibodies has also been shown to be a crucial parameter with regard to the induction of anti-idiotypic responses. For example, subcutaneous and intradermal injections have been reported to be much more immunogenic than intravenous injection (Durrant, L. G. et al., Cancer Immunol. Immunother., Vol. 28, 37-42 (1989)). It is thus important to underline that no detectable anti-idiotypic response against monoclonal antibodies developed in animal bodies implanted with cellulose sulphate encapsulated cells releasing said antibodies has been observed.
To summarize, the data presented above clearly demonstrate that implantation of encapsulated cells releasing antibodies is suitable for long-term antibody-based gene/cell therapy approaches, especially directed against cancers and viral diseases.
Accordingly, in a preferred embodiment of the invention, the encapsulated antibody-producing cells are used in therapy where a sustained release of antibodies on a long term treatment is necessary.
Such situations are for example diseases caused by chronic virus infection, such as HIV, Hepatitis B, Herpes simplex, and Herpes genitalis.
Certain, viral infections result in the exposition of specific markers or antigens at the cell surface. Such surface markers can be selectively recognised by specific antibodies which can be modulated to be toxic for the virus-infected cells. Encapsulated cells producing such antibodies can be applied for long-term treatments of viral infections.
In an other embodiment of the invention, encapsulated cells producing neutralising antibodies which recognise and bind to markers or viral receptors on the cell surface interacting with viruses in the initial phases of viral infection, providing an direct or indirect neutralising effect on the a viral infection, are provided.
Neutralising effects may in this case rely on a direct block in binding and/or cell entry of the virus and thereby prevent further infection of any target cell or indirect on lysis by the patients own complement system or on presenting the virus via antibodies to cells of the immunosystem such as monocytes or macrophages.
In a special embodiment, the invention relates to the use of the encapsulated cells according to the invention in the treatment of tumours.
In any situation in which a specific cell type, or a specific cell subset capable of self renewal turns out to be toxic or life threatening for human beings, cell specific toxic antibodies, normal or genetically modified improving its toxicity, produced by encapsulated cells for selective destruction of cells can be used for elimination of tumour cells.
An other application of the capsules according to the invention is in the treatment severe autoimmune diseases, such as Multible Sclerosis and Rheumatic Arthritis, where specific T- and B-cells responsible for the pathological effect can be constantly eliminated in long term treatments by cell-specific toxic antibodies produced by encapsulated cells
In a further embodiment of the invention, encapsulated cells producing antibodies against surface markers of parasites such as Plasmodium falciparum, P. vivax or P. malariae are provided.
Malaria is one of the three diseases that cause most deaths a year, beside TBC and HepB. The application of antibodies against Plasmodium with the consequence of marking the parasites and thereby attracting cells of the immunsystem to destroy the parasites could be used as a non toxic non harmful prophilaxis for travellers to countries with a high risk of Malaria infection.
The encapsulated, antibody-releasing cells can also be used for producing a pharmaceutical composition containing a therapeutically effective amount of the cells together with at least one pharmaceutical carrier or diluent.